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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 12, 2017 |
| Title |
PFL_E12_WT_Hoxd13VP |
| Sample type |
SRA |
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| Source name |
Forelimb
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl6CBAF1
age: E12.5
genotype: wild type
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Micro-dissected proximal and distal segments of E12.5 limbs either from wild type or mutant embryos were dissected, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed with 2% formaldehyde for 10 min at room temperature and the reaction was quenched on ice with glycine. Cells were further lysed with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1x Protease inhibitor cocktail to isolate nuclei and stored at -80°C. Nuclei from pools of at least 8 distal or proximal limbs were digested with NlaIII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Sequences ligated to fragments of interest were digested again with DpnII (New England Biolabs), ligated with T4 DNA ligase HC (Promega) in diluted conditions.
These templates were amplified using Expand long template (Roche) and inversed PCR primers flanked with adaptors allowing multiplexing. Hoxd13_inverse_forward: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTAAAATCCTAGACCTGGTCAT, Hoxd13-inverse_reverse: CAAGCAGAAGACGGCATACGAGGCCGATGGTGCTGTATAG, Isl_II forward primer: GCATTCATCAAGCTGTGATTAGCA, Isl_II reverse primer: TCCCATAATATGTAGACTGTAGTGTTGC.
For sample 9 we used a series of four different barcoded primers for Hoxd13 forward such as barcode 1: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTATCGAAAAATCCTAGACCTGGTCATG; iHoxd13 forward: barcode 2: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTGACAAAAAATCCTAGACCTGGTCATG; iHoxd13 forward: barcode3: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACAAAAATCCTAGACCTGGTCATG; and iHoxd13 forward: barcode4: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTAAAAAATCCTAGACCTGGTCATG
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Proximal limb mouse E12.5 WT_Hoxd13 viewpoint
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| Data processing |
De-multiplexing, mapping and 4C-analysis were performed through HTSstation (David et al, 2014 and http://htsstation.epfl.ch/). Reads were mapped to the mm10 (GRCm38) mouse genome assembly with bowtie version 0.12.7 (Langmead et al, 2009) with parameters “-n 2 --best -strata -m 5”. Genome coverage densities were calculated for each strand separately, then averaged and normalized by the total number of mapped reads (times 10^-7).
4C figures were made using smoothed/normalized data that were obtained by applying a running mean algorithm with a window size of eleven fragments and normalized to the same total intensity, such as to see only changes in contacts distribution and to minimize PCR complexity bias in each samples.
Genome_build: mm10
Supplementary_files_format_and_content: Smoothed/normalized data (bedGraph), one per viewpoint, per tissue
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| Submission date |
May 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Pierre J Fabre |
| E-mail(s) |
fabre.pierre@gmail.com
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| Organization name |
EPFL
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| Department |
ISREC
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| Lab |
Laboratory of Developmental Genomics
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| Street address |
Station 19
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| City |
Lausanne |
| ZIP/Postal code |
CH-1015 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE98233 |
Large-scale genomic reorganizations of topological domains (TADs) at the HoxD locus |
| GSE98861 |
Large-scale genomic reorganizations of topological domains (TADs) at the HoxD locus (4C-Seq) |
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| Relations |
| BioSample |
SAMN06952485 |
| SRA |
SRX2804237 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2616857_PFL_E12_WT_Hoxd13VP.bedgraph.gz |
3.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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